To address a critical need for improved human tissue models to study infectious diseases, we propose to form the MIT Center for Human Tissues and Infectious Diseases (MIT.HTMID). The MIT.HTMID Center will study viral infections of the human brain and central nervous system by using biologically relevant two dimensional (2D) human neural cells and three dimensional (3D) human cerebral organoids. The cells are homogeneous preparations of human neuronal progenitors, neurons, oligodendrocytes, astrocytes, and microglia, derived from embryonic stem (ES) cells or induced pluripotent stem cells (iPS cells), and will be produced in the MIT.HTMID Human Cell and Tissue Core. Prior work has demonstrated that these cells and the organoids formed are near-physiological in their biological functions, thereby representing an experimental system that is superior to rodent models. MIT.HTMID Project 2 will focus on the applied use of the 2D and 3D tissue models for the purposes of a) testing a panel of published antiviral compounds for efficacy in blocking virus infections, and b) studying the genetics of infectious disease by performing CRISPR-Cas9 gene knockouts to identify host dependency genes that regulate virus infections or are regulated by virus infections. Project 2 Aim 2A will test a panel of published antiviral compounds for efficacy in blocking virus infections. The approaches will couple the cells and organoids with infections using two types of viruses, produced in the MIT.HTMID Virology Core. First, we will study flaviviruses, including Dengue Fever Virus, West Nile Virus, and Zika Virus. Indeed, an important motivation for studying human brain tissue and viruses is to understand the pathogenesis of Zika virus infections, which are causing microcephaly and Guillain-Barre syndrome worldwide. Second, we will use pseudotyped vesicular stomatitis viruses (VSV) to study entry of several encephalitic viruses, including Eastern Equine Encephalitis Virus (EEEV), Western Equine Encephalitis Virus (WEEV) and Venezuelan Equine Encephalitis Virus (VEEV). By replacing the VSV viral envelope with that of EEEV, WEEV, or VEEV, we are able to study virus entry of select (i.e. highly dangerous with potential application in biowarfare) agents in the 2D cells and 3D organoids. Using pseudotyped select viruses and multiple relevant cell types will allow us to study virus tropism, pathology, and potential therapeutic targets. Project 2 Aim 2A is not a high throughput screen; rather, the goal is to test the efficacy of a panel of published antivirals in blocking infections of the 2D neural cells and 3D organoids. Project 2 Aim 2B.i and 2B.ii will focus on performing and validating CRISPR- Cas9 screens that couple the neural cell types with infections by Zika virus or pseudotyped VSV. These screens will identify host dependency factors (HDF) that are required for the viral replication cycle, and may identify targets for therapeutic intervention. Both the antiviral testing and the genetic screening approaches will critically evaluate the human 2D neural cells and human 3D cerebral organoids for use as a human tissue model for infectious disease.